Eggshell membrane nonapeptide and its application in preventing and treating osteoporosis

Abstract

The present invention discloses an eggshell membrane nonapeptide and its use in preventing and treating osteoporosis, belonging to the field of biotechnology. The amino acid sequence of the eggshell membrane nonapeptide is shown in SEQ ID NO. 1, specifically: Val-Pro-Pro-Gly-Asp-Tyr-Leu-Phe-Gln. The eggshell membrane nonapeptide of the present invention can repair and improve the antioxidant capacity, differentiation activity, and bone formation capacity of osteoblasts affected by oxidative stress, providing a new therapeutic approach for preventing and treating osteoporosis caused by oxidative stress.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to an eggshell membrane nonapeptide and application thereof in preventing and treating osteoporosis. Background Art

[0002] Increased bone fragility caused by osteoporosis has become a major cause of fractures in the elderly, resulting in high rates of disability and mortality, and placing a heavy burden on society. Hormonal imbalances, calcium and vitamin D deficiencies, and normal aging are generally considered the primary causes of osteoporosis. However, in recent years, oxidative stress has also begun to be identified as another important factor. Recent studies suggest that oxidative stress may be a major factor in the functional decoupling of osteoblasts and osteoclasts in osteoporosis, making regulation of oxidative stress a key approach for the treatment of osteoporosis. Although numerous drugs have been developed to prevent osteoclastic bone resorption or promote bone formation, their therapeutic efficacy is relatively poor and they have certain limitations and side effects. For example, long-term use of antiresorptive drugs can disrupt the coupling of bone resorption and formation, thereby increasing bone fragility and osteonecrosis. Therefore, developing new treatments that can reverse this imbalance in bone homeostasis while promoting osteogenesis and reducing osteoclasts is of great importance.

[0003] Eggshell membrane, a byproduct of egg processing, contains various proteins such as collagen and keratin, as well as bioactive substances such as hyaluronic acid and chondroitin sulfate, making it a potential source of bioactive peptides. However, eggshell membrane is often used as feed or landfilled, resulting in significant resource waste. Eggshell membrane has been shown to have certain antioxidant effects, making the development of bioactive peptides from eggshell membrane for the prevention and treatment of osteoporosis of great significance. Summary of the Invention

[0004] The present invention aims to provide an eggshell membrane nonapeptide and its use in preventing and treating osteoporosis to address the problems of the prior art. The eggshell membrane nonapeptide of the present invention can repair and enhance the antioxidant capacity, differentiation activity, and bone formation capacity of osteoblasts affected by oxidative stress, providing a new therapeutic approach for preventing and treating osteoporosis caused by oxidative stress.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides an eggshell membrane nonapeptide. The amino acid sequence of the eggshell membrane nonapeptide is shown in SEQ ID NO.1, specifically: Val-Pro-Pro-Gly-Asp-Tyr-Leu-Phe-Gln.

[0007] The eggshell membrane nonapeptide of the present invention can be synthesized by solid phase synthesis, liquid phase synthesis, biosynthesis or enzymatic synthesis; in some embodiments of the present invention, the eggshell membrane nonapeptide is synthesized by solid phase synthesis.

[0008] The present invention also provides the use of the eggshell membrane nonapeptide in preparing medicine for preventing and treating osteoporosis.

[0009] Preferably, the osteoporosis is osteoporosis caused by oxidative stress.

[0010] The present invention also provides use of the eggshell membrane nonapeptide in preparing a medicine for improving the antioxidant capacity of osteoblasts.

[0011] The present invention also provides use of the eggshell membrane nonapeptide in preparing a drug for promoting osteoblast differentiation.

[0012] The present invention also provides use of the eggshell membrane nonapeptide in preparing a drug for promoting osteoblast mineralization.

[0013] The present invention also provides use of the eggshell membrane nonapeptide in preparing a drug for promoting new bone formation.

[0014] The present invention also provides a medicine for preventing and treating osteoporosis, wherein the active ingredient of the medicine is the eggshell membrane nonapeptide.

[0015] Preferably, the drug further comprises a pharmaceutically acceptable carrier.

[0016] The present invention discloses the following technical effects:

[0017] The eggshell membrane nonapeptide Val-Pro-Pro-Gly-Asp-Tyr-Leu-Phe-Gln of the present invention is derived from eggshell membrane, which has a wide range of raw material sources, is highly safe, and is easily accessible. In vitro antioxidant tests and in vitro oxidative stress osteogenesis model tests have confirmed that the eggshell membrane nonapeptide has extremely high antioxidant effects, has a repairing effect on osteoblasts affected by oxidative stress, can improve the antioxidant capacity, differentiation activity, and bone formation capacity of osteoblasts, and demonstrates significant bone repair ability. This provides a new technical means for treating osteoporosis caused by oxidative stress and demonstrates excellent application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1This is the secondary mass spectrum of the peptide Val-Pro-Pro-Gly-Asp-Tyr-Leu-Phe-Gln;

[0020] Figure 2 is the ABTS and DPPH free radical scavenging rate of the peptide Val-Pro-Pro-Gly-Asp-Tyr-Leu-Phe-Gln;

[0021] Figure 3 is the activity of superoxide dismutase (T-SOD) in osteoblasts under different treatments;

[0022] Figure 4 is the alkaline phosphatase (AKP) activity of osteoblasts under different treatments;

[0023] Figure 5 Fluorescence microscopy images of osteoblast mineralization staining under different treatments;

[0024] Figure 6 SEM images of osteoblasts after mineralization under different treatments. DETAILED DESCRIPTION

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0027] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0028] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0029] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0030] The technical solutions of the present invention are as follows:

[0031] Eggshell membranes were used as raw material, and eggshell membrane hydrolysates were prepared using enzymatic hydrolysis. Peptides with molecular weights less than 3 kDa were isolated from the hydrolysate using ultrafiltration and other techniques. A highly active nonapeptide, Val-Pro-Pro-Gly-Asp-Tyr-Leu-Phe-Gln, was identified through HPLC-MS / MS, bioinformatics analysis, and AutoDock Vina molecular modeling docking. This nonapeptide was then subjected to in vitro antioxidant studies and an in vitro oxidative stress osteogenesis model to test its efficacy in osteoblasts affected by oxidative stress.

[0032] The preparation process of the eggshell membrane hydrolyzate is as follows:

[0033] 1. Take dried, purified eggshell membranes, add purified water at a material-liquid ratio of 100 g: (600-1000) mL, and then add 40 mmol / L of one or more amino acids selected from arginine, proline, serine, and cysteine ​​to the eggshell membranes to obtain an eggshell membrane suspension.

[0034] 2. Transfer the eggshell membrane suspension to an enzymatic hydrolysis tank and sequentially add keratinase (3000 U / g) and collagenase (4000 U / g) to perform a stepwise or simultaneous enzymatic hydrolysis. Keratinase reaction temperature: 30-60°C, pH: 6-10, reaction time: 1-5 hours; collagenase reaction temperature: 30-60°C, pH: 6-10, reaction time: 1-4 hours. After the reaction, heat to 80-90°C for 10-30 minutes to inactivate the enzymes and obtain the eggshell membrane hydrolyzate.

[0035] 3. Centrifuge the eggshell membrane hydrolyzate and dry it to obtain a powdered eggshell membrane hydrolyzate.

[0036] Example 1 Preparation of eggshell membrane nonapeptide

[0037] 1. Take dried and purified eggshell membranes, add purified water at a material-liquid ratio of 100 g:800 mL, and then add 40 mmol / L cysteine ​​to mix with the eggshell membranes to obtain an eggshell membrane suspension.

[0038] 2. Transfer the eggshell membrane suspension to an enzymatic hydrolysis tank and sequentially add keratinase (3000 U / g eggshell membrane substrate) and collagenase (4000 U / g eggshell membrane substrate) to perform a stepwise, combined enzymatic hydrolysis reaction. The keratinase hydrolysis reaction temperature is 50°C, pH 7.0, and the reaction time is 4 hours; the collagenase hydrolysis reaction temperature is 50°C, pH 7.0, and the reaction time is 3 hours. After the reaction, heat the mixture to 90°C for 30 minutes to inactivate the enzymes, thereby obtaining the eggshell membrane hydrolyzate.

[0039] 3. Centrifuge the eggshell membrane hydrolyzate, dry it, and extract the powdered eggshell membrane polypeptide.

[0040] 4. Screening and identification of target peptides: After dissolving the shell membrane polypeptide in sterile water, ultrafiltration and drying were performed to separate the polypeptide components with a molecular weight of less than 3K Da. After preparing the polypeptide solution, HPLC-MS / MS identification and bioinformatics analysis were performed. The peptide property calculator online tool was used to predict the function of the peptide. Subsequently, molecular simulation docking was performed using AutoDockVina to identify a highly active nonapeptide Val-Pro-Pro-Gly-Asp-Tyr-Leu-Phe-Gln (SEQ ID NO. 1). The secondary mass spectrum of the nonapeptide is shown in the figure below. Figure 1 shown.

[0041] Example 2 Functional Verification of Eggshell Membrane Nonapeptide

[0042] The nonapeptide Val-Pro-Pro-Gly-Asp-Tyr-Leu-Phe-Gln selected in Example 1 was synthesized by solid-phase synthesis, and the function of the nonapeptide was verified. The process is as follows:

[0043] 1. In vitro antioxidant test: The nonapeptide was prepared into a peptide solution with a concentration of 1 mg / mL, and the free radical scavenging rate was measured in ABTS and DPPH reagents. The results are as follows Figure 2 As shown in the results, the ABTS free radical scavenging rate of this peptide can reach more than 80%, and the DPPH free radical scavenging rate can reach more than 85%.

[0044] 2. Intracellular Activity Evaluation Experiment: First, mouse embryonic osteoblasts MC3T3-E1 (purchased from ATCC) were treated with 400 mmol / L H2O2 for 24 hours to establish an oxidative stress osteoblast model. Subsequently, the oxidative stress osteoblasts were treated with complete culture medium containing 100 μg / mL of the aforementioned peptide solution as the nonapeptide group (VPPGDYLFQ group), and those treated with complete culture medium without the aforementioned peptide solution as the model group (Model group). Normal MC3T3-E1 cells served as the control (Control group). The superoxide dismutase (T-SOD) and alkaline phosphatase (AKP) activities of the cells in each group were measured.

[0045] The results of T-SOD activity test were as follows: Figure 3 As shown in the results, the T-SOD activity of osteoblasts treated with H2O2 decreased significantly compared with normal cells, indicating that the oxidative stress osteoblast model was successfully established. Compared with the model group, the nonapeptide group significantly improved the decrease in T-SOD activity caused by oxidative stress, indicating that the nonapeptide has a certain antioxidant repair ability in cells.

[0046] AKP activity test results are as follows Figure 4 As shown in the results, the osteoblasts treated with H2O2 showed a significant decrease in AKP activity compared with normal cells. Compared with the model group and the control group, the nonapeptide group significantly increased AKP activity, indicating that it can alleviate the inhibitory effect of oxidative stress on the differentiation function of osteoblasts.

[0047] 3. Mineralized Nodule Staining Image: Osteoblasts are the primary functional cells responsible for bone formation, responsible for new bone formation. The extracellular matrix secreted by osteoblasts can produce mineralization. Mineralized nodules are a hallmark of osteoblast differentiation and maturation, and are also the primary morphological characteristic of osteoblasts performing their bone-forming function. Observing mineralized nodules in osteoblasts is a common method for studying osteoblast differentiation. Calcium salts produced by osteoblasts cultured in vitro bind to alizarin red to form a stable red pigment complex, which appears red.

[0048] The cells in the model group and the nonapeptide group cultured for 21 days were washed three times with PBS and fixed with 4% paraformaldehyde for 1 hour. The fixative was discarded, and after washing with PBS, 1 mL of 0.1% Alizarin Red working solution was added to each well of the 6-well plate and stained for 15 minutes. The staining solution was discarded, and the stained cells were rinsed with deionized water on a shaker five times for 3 minutes each time. The cells were dried in a 37°C incubator overnight, and the stained cells were scanned and imaged.

[0049] The results are as follows Figure 5As shown, the osteoblasts in the nonapeptide group produced obvious mineralized nodules, and the osteoblasts had begun to play a role in new bone synthesis, while the model group had almost no formation of mineralized nodules, which showed that the new bone synthesis of osteoblasts was inhibited by oxidative stress, and the nonapeptide of the present invention can improve the differentiation ability of osteoblasts by regulating oxidative stress, indicating that to a certain extent, the nonapeptide can alleviate the differentiation function damage of oxidative stress-type osteoblasts and promote the formation of their mineralized nodules.

[0050] 4. Scanning electron microscopy: The osteoblasts that produced mineralized nodules in the model group and the nonapeptide group were washed three times with ultrapure water, freeze-dried, and gold-coated. They were placed in a scanning electron microscope chamber and vacuumed. The accelerating voltage was set to 15 kV, and the structure of the osteoblast mineralized nodules under different treatments was observed under a JSM.6390 / LV scanning electron microscope. The results are as follows: Figure 6 As shown, the osteoblast mineralization nodules in the model group were smaller, while after culture with the nonapeptide, the cell mineralization nodules enlarged and showed an accumulation state, indicating that it can effectively promote the biomineralization of osteoblasts.

[0051] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. An eggshell membrane nonapeptide, characterized in that The amino acid sequence of the eggshell membrane nonapeptide is VPPGDYLFQ.

2. Use of the eggshell membrane nonapeptide according to claim 1 in the preparation of a medicament for preventing and treating osteoporosis.

3. The use according to claim 2, characterized in that The osteoporosis is osteoporosis caused by oxidative stress.

4. The use according to claim 2 or 3, characterized in that The medicine prevents and treats osteoporosis by improving the antioxidant capacity of osteoblasts.

5. The use according to claim 2 or 3, characterized in that The drug prevents and treats osteoporosis by promoting osteoblast differentiation.

6. The use according to claim 2 or 3, characterized in that The drug prevents and treats osteoporosis by promoting osteoblast mineralization.

7. The use according to claim 2 or 3, characterized in that The drug prevents and treats osteoporosis by promoting new bone formation.

8. A drug for preventing and treating osteoporosis, characterized in that: The active ingredient of the medicine is the eggshell membrane nonapeptide according to claim 1.

9. The drug according to claim 8, characterized in that The medicine also includes a pharmaceutically acceptable carrier.

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